Urinary bladder cancer (BCa) is the fourth/tenth most common cancer among men/women in the United States. It is reported that males have significantly (approximately three times) higher risk of bladder cancer than females. However, the etiology of this sex difference in incidence is unknown. Recent studies have suggested the involvement of androgens/androgen receptor (AR) signaling in BCa progression. Using AR knockout (ARKO) mice, we found that the androgen/AR signaling might play a critical role in the development of the chemical carcinogen, N-butyl-N-4-hydroxybutyl-nitrosamine (BBN), induced bladder carcinogenesis. With BBN treatment, ARKO mice, that lack AR and with undetectable androgen, did not develop bladder tumors while their wild type littermates, with functional AR, all develop bladder tumors at the age of 50 wks. Interestingly and unexpectedly, we found near 25% of ARKO mice develop BBN-induced bladder tumor after supplementation with the androgen, dihydrotestosterone (DHT), suggesting DHT is able to function through non-AR pathways to promote bladder tumor progression. This finding was further confirmed in the human AR-negative bladder cancer cell line, TCC5637, showing that addition of DHT can promote this AR-negative bladder cell growth and invasion. Preliminary data also showed DHT could enhance the pathways from G-proteins signals to ERK/MAPK-MEK signals in BCa AR- negative cells. Based on these in vitro human cell line and in vivo mice evidences, we hypothesize that DHT can function through non-AR pathways to promote BCa initiation, growth, and invasion. We will apply the following 4 aims to prove our hypothesis and dissect potential mechanisms. Aim 1: Using the Uro- SV40T-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 2: Using UPII-Ha-ras-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 3: Using cell transformation/tumorigenesis assays to prove DHT can go through non-AR mediated pathways to promote BCa initiation and/or progression. Aim 4: Using human AR-negative bladder cell lines to prove DHT can go through non-AR pathways to promote BCa growth and invasion and to dissect potential mechanisms. IMPACT: Currently, BCa in humans is not generally considered hormonally dependent and therefore it is not assumed that androgen deprivation therapy (ADT) with chemical or surgical castration can be an effective treatment option for BCa patients in order to repress tumor progression. The success of this proposal to prove DHT can function through non-AR pathways to promote bladder cancer initiation and progression, not only provides in vivo evidence for a novel androgen mechanism, it may also answer the puzzle for the previous failure of using ADT to suppress androgen binding to AR for the treatment of BCa patients and may provide a new preventive/therapeutic approach to suppress BCa.